[Show abstract][Hide abstract] ABSTRACT: The human copper chaperone Atox1 plays a central role in the transport of copper in cells. It has been reported that the conserved residue Lys60 contributes to the heterocomplex stability of Atox1 with its target protein ATPase, and that the K60A mutation could diminish the copper transfer. In this work, we carried out the structure determination and dynamic analysis of Atox1 with the K60A mutation in order to elucidate the role of the conserved residue Lys60 in the copper transport. Results show that the K60A mutation results in crucial secondary structure rearrangements and side-chain orientation alteration of the metal-binding residues in Atox1. Protein dynamic studies reveal that the K60A mutation leads to increased overall flexibility, and a significant difference in dynamic properties of the metal-binding sites. The structure and dynamic changes cause a decrease in the copper-binding stability of the K60A mutant. In addition, Cu(i)-mediated hetero-protein interactions with ATP7A are present in the metal transfer of both Atox1 variants, although copper transfer is accompanied with smaller structural alteration in the K60A mutant. These results indicate that Lys60 is crucial in maintaining the structure and dynamic properties of Atox1.
[Show abstract][Hide abstract] ABSTRACT: The FANCM/FAAP24 heterodimer has distinct functions in protecting cells from complex DNA lesions such as interstrand crosslinks. These functions rely on the biochemical activity of FANCM/FAAP24 to recognize and bind to damaged DNA or stalled replication forks. However, the DNA-binding activity of this complex was not clearly defined. We investigated how FAAP24 contributes to the DNA-interacting functions of the FANCM/FAAP24 complex by acquiring the N-terminal and C-terminal solution structures of human FAAP24. Modeling of the FAAP24 structure indicates that FAAP24 may possess a high affinity toward single-stranded DNA (ssDNA). Testing of various FAAP24 mutations in vitro and in vivo validated this prediction derived from structural analyses. We found that the DNA-binding and FANCM-interacting functions of FAAP24, although both require the C-terminal (HhH)2 domain, can be distinguished by segregation-of-function mutations. These results demonstrate dual roles of FAAP24 in DNA damage response against crosslinking lesions, one through the formation of FANCM/FAAP24 heterodimer and the other via its ssDNA-binding activity required in optimized checkpoint activation.Cell Research advance online publication 3 September 2013; doi:10.1038/cr.2013.124.
[Show abstract][Hide abstract] ABSTRACT: Rv0899 from Mycobacterium tuberculosis belongs to the OmpA (outer membrane protein A) family of outer membrane proteins. It functions as a pore-forming protein; the deletion of this gene impairs the uptake of some water-soluble substances, such as serine, glucose, and glycerol. Rv0899 has also been shown to play a part in low-pH environment adaption, which may play a part in pathogenic mycobacteria overcoming the host's defense mechanisms. Based on many bacterial physiological data and recent structural studies, it was proposed that Rv0899 forms an oligomeric channel to carry out such functions. In this work, biochemical and structural data obtained from solution NMR and EPR spectroscopy indicated that Rv0899 is a monomeric membrane-anchoring protein with two separate domains, rather than an oligomeric pore. Using NMR chemical shift perturbation and isothermal calorimetric titration assays, we show that Rv0899 was able to interact with Zn(2+) ions, which may indicate a role for Rv0899 in the process of Zn(2+) acquisition.
Journal of Molecular Biology 11/2011; 415(2):382-92. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Site-specific ¹⁹F chemical shift and side chain relaxation analysis can be applied on large size proteins. Here, one-dimensional ¹⁹F spectra and T₁, T₂ relaxation data were acquired on a SH3 domain in aqueous buffer containing 60% glycerol, and a nine-transmembrane helices membrane protein diacyl-glycerol kinase (DAGK) in dodecyl phosphochoine (DPC) micelles. The high quality of the data indicates that this method can be applied to site-specifically analyze side chain internal mobility of membrane proteins or large size proteins.
Protein Science 11/2010; 20(1):224-8. · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for (15)N/(19)F-trifluoromethyl-phenylalanine ((15)N/(19)F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide ((15)N)(1)H and side-chain (19)F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide ((15)N)(1)H and side-chain (19)F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes.
Biochemical and Biophysical Research Communications 10/2010; 402(3):461-6. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Zinc finger motif contains a zinc ion coordinated by several conserved amino acid residues. Yeast Sgf73 protein was identified as a component of SAGA (Spt/Ada/Gcn5 acetyltransferase) multi-subunit complex and Sgf73 protein was known to contain two zinc finger motifs. Sgf73(1-104), containing the first zinc finger motif, was necessary to modulate the deubiquitinase activity of SAGA complex. Here, Sgf73(1-104) was over-expressed using bacterial expression system and purified for solution NMR (nuclear magnetic resonance) structural studies. Secondary structure and site-specific relaxation analysis of Sgf73(1-104) were achieved after solution NMR backbone assignment. Solution NMR and circular dichroism analysis of Sgf73(1-104) after zinc ion removal using chelation reagent EDTA (ethylene-diamine-tetraacetic acid) demonstrated that zinc ion was required to maintain stable conformation of the zinc finger motif.
Biochemical and Biophysical Research Communications 07/2010; 397(3):436-40. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inwardly rectifying potassium channel 2.3 (Kir2.3) is specifically targeted on the basolateral membranes of epithelial and neuronal cells, and it thus plays an important role in maintaining potassium homeostasis. Tax-interacting protein-1 (TIP-1), an atypical PDZ-domain-containing protein, binds to Kir2.3 with a high affinity, causing the intracellular accumulation of Kir2.3 in cultured epithelial cells. However, the molecular basis of the TIP-1/Kir2.3 interaction is still poorly understood. Here, we present the crystal structure of TIP-1 in complex with the C-terminal Kir2.3-peptide (residues 436-445) to reveal the molecular details of the interaction between them. Moreover, isothermal titration calorimetry experiments show that the C-terminal Kir2.3-peptide binds much more strongly to TIP-1 than to mammalian Lin-7, indicating that TIP-1 can compete with mammalian Lin-7 to uncouple Kir2.3 from its basolateral membrane anchoring complex. We further show that the phosphorylation/dephosphorylation of Ser443 within the C-terminal Kir2.3 PDZ-binding motif RRESAI dynamically regulates the Kir2.3/TIP-1 association in heterologous HEK293T cells. These data suggest that TIP-1 may act as an important regulator for the endocytic pathway of Kir2.3.
Journal of Molecular Biology 08/2009; 392(4):967-76. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In membrane protein biochemical and structural studies, detergents are used to mimic membrane environment and maintain functional, stable conformation of membrane proteins in the absence of lipid bilayers. However, detergent concentration, esp. molar ratio of membrane protein to detergent is usually unknown. Here, a gas chromatography-mass spectrometry selected ion monitoring (GC-MS-SIM) method was developed to quantify four detergents which are frequently used in membrane protein structural studies. To remove excessive detergents, a filtered centrifugation using Centricon tubes was applied. A membrane protein Ig-Beta fragment in four different detergent micelles was exemplified. Detergent concentrations in the upper and lower fraction of the Centricon tube were measured after each round of centrifugation. The results were very consistent to basic properties of detergent micelles in aqueous solvents. Therefore, coupling of GC-MS-SIM and detergent removal by Centricon tubes, detergents concentration, esp. molar ratio of membrane protein to detergent could be controlled, which will expedite membrane protein structural and biochemical studies.
Protein Expression and Purification 08/2009; 68(2):221-5. · 1.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel and reliable gas chromatography-flame ionization detection (GC-FID) method that can separate and quantify detergents frequently used in membrane protein structural studies has been developed. Different detergents were identified through FID peaks with different retention times. A quadratic regression curve was found to fit the integrated FID peak area against different detergent concentrations. Detergents can be quantified as low as the nanogram level: lauryl-dimethylamine-N-oxide (LDAO), 5 ng; dodecyl maltoside (DDM), 10 ng; and dodecyl phosphocholine (DPC), 50 ng. This method can be applied directly to measure detergent concentration and molar ratio of membrane protein to detergents during membrane protein extraction, purification, concentration, and crystallization.
[Show abstract][Hide abstract] ABSTRACT: Tax-interacting protein-1 (TIP-1) is an unusual signaling protein, containing a single PDZ domain. TIP-1 is able to bind beta-catenin with high affinity and thus inhibit its transcriptional activity. The high-resolution crystal structure of TIP-1 in complex with the C-terminal peptide of beta-catenin provides molecular details for the recognition of beta-catenin by TIP-1. Moreover, structural comparison of peptide-free and peptide-bound TIP-1 reveals that significant conformational changes are required in the betaB-betaC loop region of TIP-1 to avoid clashes with the incoming C-terminal beta-catenin peptide. Such conformational changes have not been observed in other structures of PDZ domains. In addition to the canonical peptide-binding pocket of the PDZ domain, TIP-1 can form a binding cavity to anchor more amino acids through a conserved hydrophobic residue pair (Trp776 of beta-catenin and Pro45 of TIP-1). Structural and biochemical data indicate that the canonical binding pocket together with the hydrophobic residue pair are presumably the major cause of the significantly higher affinity of the beta-catenin C-terminal to TIP-1 than to other PDZ domains, providing a unique binding specificity. Our results reveal the molecular mechanism of TIP-1 as an antagonist in PDZ domain signaling.
Journal of Molecular Biology 10/2008; 384(1):255-63. · 3.91 Impact Factor